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PubMed2006

Enterobacterial repetitive intergenic consensus (ERIC) sequences in Escherichia coli: Evolution and implications for ERIC-PCR.

Enterobacterial repetitive intergenic consensus (ERIC) sequences are 127-bp imperfect palindromes that occur in multiple copies in the genomes of enteric bacteria and vibrios. Here we investigate the distribution of these elements in the complete genome sequences of nine Escherichia coli (including Shigella species) strains. There is a significant tendency for copies to be adjacent to more highly expressed genes. There is considerable variation among strains with respect to the presence of an element in any particular intergenic region, but some copies appear to have been conserved since before the divergence of E. coli and Salmonella enterica. In comparisons of orthologous copies between these species, ERIC sequences are surprisingly conserved, implying that they have acquired some function, perhaps related to mRNA stability. The relationships among copies within E. coli are consistent with a master copy mode of generation. Insertion of new copies seems to occur at, and involve duplication of, the dinucleotide TA. Two classes of inserts of about 70 bp each occur at different specific sites within ERIC sequences; these inserts evolve independently of the ERIC sequences. The small number of ERIC sequences in E. coli genomes indicates that a widely used bacterial fingerprinting method using primers based on ERIC sequences (ERIC-PCR) does not rely on the presence of ERIC sequences.

Base Sequence↗

PubMed2001

Contrasting levels of nucleotide diversity on the avian Z and W sex chromosomes.

Sex chromosomes may provide a context for studying the local effects of mutation rate on molecular evolution, since the two types of sex chromosomes are generally exposed to different mutational environments in male and female germ lines. Importantly, recent studies of some vertebrates have provided evidence for a higher mutation rate among males than among females. Thus, in birds, the Z chromosome, which spends two thirds of its time in the male germ line, is exposed to more mutations than the female-specific W chromosome. We show here that levels of nucleotide diversity are drastically higher on the avian Z chromosome than in paralogous sequences on the W chromosome. In fact, no intraspecific polymorphism whatsoever was seen in about 3.4 kb of CHD1W intron sequence from a total of >150 W chromosome copies of seven different bird species. In contrast, the amount of genetic variability in paralogous sequences on the Z chromosome was significant, with an average pairwise nucleotide diversity (d) of 0.0020 between CHD1Z introns and with 37 segregating sites in a total of 3.8 kb of Z sequence. The contrasting levels of genetic variability on the avian sex chromosomes are thus in a direction predicted from a male-biased mutation rate. However, although a low gene number, as well as some other factors, argues against background selection and/or selective sweeps shaping the genetic variability of the avian W chromosome, we cannot completely exclude selection as a contributor to the low levels of variation on the W chromosome.

Animals↗

PubMed2004

An RT-PCR-based strategy to estimate full-length CYP2D6 mRNA copy number.

The goal of this study is to develop an analytical tool to assess cytochrome P450 2D6 (CYP2D6) levels in the form of full-length transcripts. CYP2D6 RNA in test samples was evaluated by co-amplification with an internal RNA control in a reverse-transcribed polymerase chain reaction (RT-PCR). The internal CYP2D6 RNA control was constructed by internally deleting 474 bp of CYP2D6 RNA, allowing simultaneous amplification of the test RNA together with the internal control RNA in a single RT-PCR reaction. With sequential dilution of test RNA, the CYP2D6 mRNA transcript levels in test samples were estimated. The full-length RT-PCR strategy allowed semiquantitative assessments of CYP2D6 RNA transcripts with a sensitivity limit of 500 copies for CYP2D6 RNA transcripts, 2500 copies/microg total human liver RNA, and 10% intraday coefficient of variation (CV). In a method validation study, the CYP2D6 activity appeared to relate more closely to full-length CYP2D6 mRNA concentration than a short-sequence of CYP2D6 RNA estimated with a real-time quantitative RT-PCR assay. We have developed an efficient semiquantitative assay and demonstrated its suitability for estimating full-length CYP2D6 mRNA transcripts in cells and tissues.

Cytochrome P-450 CYP2D6↗

PubMed2026

A Second Report of a Missense Variant in AMMECR1 Causing Midface Hypoplasia, Hearing Impairment, Elliptocytosis, and Nephrocalcinosis: Case Report and Literature Review.

Pathogenic variants in AMMECR1 have been associated with a rare multisystem disorder characterized by midface hypoplasia, hearing impairment, elliptocytosis, and nephrocalcinosis (MFHIEN). To date, most reported cases involve copy number variants or presumed loss-of-function alterations, with only a single prior report describing a missense variant supported by functional studies. Here, we report a patient with a heterozygous de novo AMMECR1 missense variant, NM_015365.3:c.649G>A p.(Val217Met) presenting with clinical features consistent with MFHIEN, including midface hypoplasia, partial hearing impairment, nephrocalcinosis, and elliptocytosis identified on peripheral blood smear. Comparative review of the literature highlights that while previously reported missense variants in AMMECR1 demonstrated altered intranuclear protein distribution and reduced expression in functional assays, clinical evidence supporting pathogenicity of non-truncating variants remains limited. The phenotypic overlap between our patient and prior report strengthens the association between missense variations and the MFHIEN phenotype. Our findings support the pathogenic relevance of missense variation in AMMECR1 and emphasize the importance of integrating detailed phenotyping, including hematologic evaluation, with genomic data in the diagnosis of rare multisystem disorders. Additional cases and functional studies are needed to clarify genotype-phenotype correlations and underlying disease mechanisms.

Humans↗

PubMed2006

Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.

Base Sequence↗

PubMed2006

PRKCA and multiple sclerosis: association in two independent populations.

Multiple sclerosis (MS) is a chronic disease of the central nervous system responsible for a large portion of neurological disabilities in young adults. Similar to what occurs in numerous complex diseases, both unknown environmental factors and genetic predisposition are required to generate MS. We ascertained a set of 63 Finnish MS families, originating from a high-risk region of the country, to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24. Initial single nucleotide polymorphism (SNP)-based association implicated PRKCA (protein kinase C alpha) gene, and this association was replicated in an independent set of 148 Finnish MS families (p = 0.0004; remaining significant after correction for multiple testing). Further, a dense set of 211 SNPs evenly covering the PRKCA gene and the flanking regions was selected from the dbSNP database and analyzed in two large, independent MS cohorts: in 211 Finnish and 554 Canadian MS families. A multipoint SNP analysis indicated linkage to PRKCA and its telomeric flanking region in both populations, and SNP haplotype and genotype combination analyses revealed an allelic variant of PRKCA, which covers the region between introns 3 and 8, to be over-represented in Finnish MS cases (odds ratio = 1.34, 95% confidence interval 1.07-1.68). A second allelic variant, covering the same region of the PRKCA gene, showed somewhat stronger evidence for association in the Canadian families (odds ratio = 1.64, 95% confidence interval 1.39-1.94). Initial functional relevance for disease predisposition was suggested by the expression analysis: The transcript levels of PRKCA showed correlation with the copy number of the Finnish and Canadian "risk" haplotypes in CD4-negative mononuclear cells of five Finnish multiplex families and in lymphoblast cell lines of 11 Centre d'Etude du Polymorphisme Humain (CEPH) individuals of European origin.

Adult↗

PubMed2001

Dynamics of R1 and R2 elements in the rDNA locus of Drosophila simulans.

The mobile elements R1 and R2 insert specifically into the rRNA gene locus (rDNA locus) of arthropods, a locus known to undergo concerted evolution, the recombinational processes that preserve the sequence homogeneity of all repeats. To monitor how rapidly individual R1 and R2 insertions are turned over in the rDNA locus by these processes, we have taken advantage of the many 5' truncation variants that are generated during the target-primed reverse transcription mechanism used by these non-LTR retrotransposons for their integration. A simple PCR assay was designed to reveal the pattern of the 5' variants present in the rDNA loci of individual X chromosomes in a population of Drosophila simulans. Each rDNA locus in this population was found to have a large, unique collection of 5' variants. Each variant was present at low copy number, usually one copy per chromosome, and was seldom distributed to other chromosomes in the population. The failure of these variants to spread to other units in the same rDNA locus suggests a strong recombinational bias against R1 and R2 that results in the individual copies of these elements being rapidly lost from the rDNA locus. This bias suggests a significantly higher frequency of R1 and R2 retrotransposition than we have previously suggested.

Animals↗

PubMed2005

Role of poly(A) tail length in Alu retrotransposition.

Alu are mobile noncoding Short INterspersed Elements (SINEs) present at a million copies in the human genome. Using marked Alu sequences in an ex vivo assay, we previously showed that they are mobilized through diversion of the LINE (Long INterspersed Elements) retrotransposition machinery, with the poly(A) tail of the Alu being required for their mobility. Here we show that other homopolymeric tracts cannot functionally replace the Alu poly(A) tail, and that the Alu transposition rate varies over a two-log range depending on the poly(A) tail length. Variation is according to a sigmoid-shaped curve with a lag observed for tails shorter than 15 nt and a plateau reached for tails longer than 50 nt, consistent with the binding of a limited number of a protein component requiring multiple contacts for a productive interaction with the poly(A) stretch. This analysis indicates that most of the naturally occurring genomic Alu, owing to their pA tail length, should be poor substrates for the LINE machinery, a feature possibly "selected" for the host sake.

Alu Elements↗

PubMed1998

Gene sequence and transcription differences in 70 kDa heat shock protein correlate with murine virulence of Toxoplasma gondii.

This study compared the genes encoding cytoplasmic 70 kDa heat shock protein in virulent and avirulent strains of Toxoplasma gondii, to determine whether differences may contribute to the variation in protein expression levels previously reported for this protozoan parasite. A T. gondii PCR probe with homology to Eimeria acervulina cytoplasmic 70 kDa heat shock protein was used to screen a genomic DNA mini-library and isolate the gene from the virulent RH strain. The entire coding region was subsequently amplified from the avirulent ME49 strain by PCR. Alignment of the gene sequences revealed that the virulent RH strain had four copies of a seven-aa repeat unit (GGMPGGM) at the 3'- end of the gene compared with five copies in the avirulent ME49 strain. Comparison of this region among other virulent and avirulent strains revealed that this difference was consistent with virulence. Copy number estimation revealed that this gene is single-copy in both the RH and the ME49 strains. Analysis of mRNA expression revealed a 1.5- to 2-fold increase in transcription of this gene in virulent strains when compared with avirulent strains. For each strain, mRNA was observed at similar levels whether grown in vivo or in vitro. Also, heat-shock treatment of tachyzoites prior to harvest did increase mRNA levels in vitro. This suggests that post-transcriptional regulation of cytoplasmic 70 kDa heat shock protein may occur in T. gondii. Differences in cytoplasmic 70 kDa heat shock protein have been demonstrated at genomic and transcriptional levels in virulent strains compared with avirulent strains, suggesting that this 70 kDa heat shock protein may play an important role in the virulence of T. gondii.

Amino Acid Sequence↗

PubMed1999

Quantitation of erbB-2 gene copy number in breast cancer by an improved polymerase chain reaction (PCR) technique, competitively differential PCR.

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and a reference gene in breast cancers. The method, termed 'competitively differential polymerase chain reaction' (CD-PCR), combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR (D-PCR) and competitive PCR (C-PCR). The CD-PCR methodology was evaluated for sensitivity and specificity by comparing amplification measured by CD-PCR with that obtained by fluorescence in situ hybridization (FISH), C-PCR, and Southern blotting analysis. CD-PCR analysis proved to be an accurate predictor of amplification. CD-PCR also overcomes the problems involved in variation of PCR efficiencies and DNA concentrations in tumor samples, and the problems caused by the plateau effect in PCR.

Base Sequence↗

PubMed1985

Transition stages of molecular drive in multiple-copy DNA families in Drosophila.

Multigene and non-genic DNA families are in a state of turnover and hence are continually being replaced throughout a population by new variant repeats. To quantify such molecular processes, in the absence of selection, it is necessary to find and compare stages of transistion during the homogenization of at least two non-genic families evolving in parallel in a closely related group of species. Detailed sequence analysis of patterns of variation, at each nucleotide position considered independently, amongst repeats of two tandem DNA families from seven related Drosophila species, reveals all stages of transition during the spread of randomly produced variant repeats. Variant repeats are found at different stages of homogenization and fixation in a population, irrespective of the loci, chromosomes or individuals from which they were cloned. Differences between the families in the relatively small number of variants at each transition stage and the greater number of fully homogenized and fixed variants between species of greater divergence indicate that the process of spread (molecular drive) is rapid relative to the mutation rate and occurs at seemingly different constant rates for each family. Occasional gene conversions, in addition to unequal exchanges, have contributed to family turnover. The significance of these results to the evolution of functional multigene families and divergence and conservation of sequences is discussed.

Journal Article↗

PubMed2000

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans.

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.

Amino Acid Sequence↗

PubMed2004

In situ chromosomal localization of rDNA sites in "Safed Musli" Chlorophytum ker-gawl and their physical measurement by fiber FISH.

Fluorescence In Situ Hybridization (FISH) technique has been applied on somatic chromosomes and extended DNA fibers in the medicinally important species of Chlorophytum to elucidate physical localization and measurement of the rDNA sites using two rRNA multigene families homologous to 45S and 5S rDNA. The two species of Chlorophytum, namely C. borivillianum and C. comosum, both with 2n = 28, reveal diversity for copy number and localization of rDNA sites. C. borivillianum is comprised of five 45S-rDNA sites:one each in the secondary constriction region of chromosomes 7, 8, 9; one in the subtelomeric region of the short arm of chromosome 2 and the telomeric region of the short arm of chromosome 12; and one 5S-rDNA site in the subtelomeric region of the long arm of chromosome 1. In C. comosum, there are three 45S-rDNA sites (one each in the short arm of chromosomes 12, 13, and 14) and two 5S-rDNA sites (in the secondary constriction regions of chromosomes 2 and 13). Fiber FISH analysis conducted on extended DNA fibers revealed variation in the size of continuous tandem strings for the two r-DNA families. Taking the standard value of native B DNA equivalent to 3.27 kb for 1 mum, it was estimated that the physical size of continuous DNA strings is of the order of approximately 90 kb, 180 kb, and 300 kb for 45S-rDNA and of the order of 60 kb, 150 kb for 5S-rDNA in C. comosum, grossly in correspondence to their respective physical sizes at metaphase.

Asparagaceae↗

PubMed1994

Properties and engineering of a mutant STA promoter of Saccharomyces diastaticus.

A new allelic variant of the STA2 gene of S. diastaticus, designated as STA2K, was cloned and characterized (1; accompanying paper). An application-oriented analysis of the promoter region of STA2K is described, with an emphasis on its peculiar structural feature: A 1.1-kb natural deletion located 189 nucleotides upstream of the translation start codon. The strength of the STA2K promoter was found comparable to that of known strong constitutive yeast promoters (ADH1, GAPDH). Regulated glucoamylase expression was demonstrated by chimeric promoters, which were constructed by placing the STA2K promoter under the control of either the PHO5 or CYC1 upstream regulatory sequences. On high-copy-number vectors, induction of the UASPHO5-STA2K chimeric promoter by phosphate depletion resulted in a destructive overexpression of the secreted glucoamylase, which completely halted cell growth, and promoted cell decay. In contrast, UASCYC1 was shown to mediate a fine-tuned regulation both by glucose concentration and, indirectly, by starch, the substrate for the glucoamylase to produce glucose.

Base Sequence↗

PubMed1997

Organisation and molecular analysis of repeated DNA sequences in the rice blast fungus Magnaporthe grisea.

The distribution of a previously described repeated DNA sequence present as a 1.3-kb PstI fragment in the genome of the rice blast fungus Magnaporthe grisea was analysed by carrying out DNA fingerprint analysis of 36 isolates including rice, non-rice and laboratory strains. The analysis of various higher-molecular-weight PstI fragments with homology to the 1.3-kb repeat revealed that these may arise predominantly from transposon insertions or point mutations. Analysis of a 5.1-kb derivative revealed both a point mutation at a PstI site and an insertion of a putative transposable element which caused an increase in molecular weight from 1.3 to 5.1 kb. Another repeat element of 1.4 kb was identified and found to exist in association with the 1.3-kb repeat. Both 1.3- and 1.4-kb elements were found to be parts of MGR583 (Hamer et al. 1989), a LINE-like element. These elements were present in a high copy number in all the rice and a majority of non-rice pathogens indicating that MGR583 is not a host-specific sequence as reported earlier. Our results suggest that repeated DNA elements in M. grisea have amplified independently of one another and further indicate that different isolates of M. grisea may have evolved from several distinct lines of origin.

Ascomycota↗

PubMed2004

Porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes.

Type I interferons (IFN-alpha and -beta) play an important role in the innate host defense against viral infection by inducing antiviral responses. In addition to direct antiviral activities, type I IFN serves as an important link between the innate and adaptive immune response through multiple mechanisms. Therefore, the outcome of a viral infection can be affected by IFN induction and the IFN sensitivity of a virus. North American porcine reproductive and respiratory syndrome virus (PRRSV) field isolates were studied with regard to IFN-alpha sensitivity and induction in order to understand the role of type I IFN in PRRSV pathogenesis. PRRSV isolates were differentially sensitive to porcine recombinant IFN-alpha (rIFN-alpha) and varied in their ability to induce IFN-alpha in porcine alveolar macrophages (PAM) cultures as measured by a porcine IFN-alpha specific ELISA on cell culture supernatants. Fifty-two plaques were purified from three PRRSV isolates (numbers 3, 7, and 12) and tested for IFN sensitivity and IFN induction. Plaque-derived populations were composed of heterogeneous populations in terms of IFN-inducing capacity and sensitivity to rIFN-alpha. When macrophages infected with isolates 3, 7, or 12 were treated with polycytidylic acid (polyI:C), IFN-alpha production was enhanced. Cells infected with isolate 3 and treated with polyI:C showed the most consistent and strongest enhancement of IFN-alpha production. It was demonstrated that the relatively low concentrations of IFN-alpha produced by isolate 3 contributed to the enhanced IFN-alpha synthesis in response to polyI:C. Isolates 7 and 12 significantly suppressed the enhanced IFN-alpha production by isolate 3 in polyI:C treated cells. To determine if suppression was at the level of IFN-alpha transcription, quantitative RT-PCR was performed for IFN-alpha mRNA and compared to GAPDH and cyclophilin mRNA quantification. However, the relative number of IFN-alpha transcript copies did not correlate with IFN-alpha protein levels, suggesting a post-transcriptional mechanism of suppression. In summary, these results demonstrate that PRRSV field isolates differ both in IFN-alpha sensitivity and induction. Furthermore, a PRRSV field isolate strongly enhance polyI:C-induced IFN-alpha production in PAM cultures and this priming effect was suppressed by other PRRSV isolates.

Animals↗

PubMed2006

Ribosomal DNA in the grasshopper Podisma pedestris: escape from concerted evolution.

Eukaryote nuclear ribosomal DNA (rDNA) typically exhibits strong concerted evolution: a pattern in which several hundred rDNA sequences within any one species show little or no genetic diversity, whereas the sequences of different species diverge. We report a markedly different pattern in the genome of the grasshopper Podisma pedestris. Single individuals contain several highly divergent ribosomal DNA groups. Analysis of the magnitude of divergence indicates that these groups have coexisted in the Podisma lineage for at least 11 million years. There are two putatively functional groups, each estimated to be at least 4 million years old, and several pseudogene groups, many of which are transcribed. Southern hybridization and real-time PCR experiments show that only one of the putatively functional types occurs at high copy number. However, this group is scarcely amplified under standard PCR conditions, which means that phylogenetic inference on the basis of standard PCR would be severely distorted. The analysis suggests that concerted evolution has been remarkably ineffective in P. pedestris. We propose that this outcome may be related to the species' exceptionally large genome and the associated low rate of deletion per base pair, which may allow pseudogenes to persist.

Animals↗

PubMed1991

Human T-cell receptor V alpha gene polymorphism.

Polymorphism in the germline repertoire of T-cell receptor (TCR) variable alpha and beta (V alpha and V beta) genes could alter the relative abilities of individuals in a population to respond to particular antigens. Variation in the number of germline V alpha and V beta gene segments has been reported in wild mice and in different inbred mouse strains. A previous study of the human V beta gene germline repertoire failed to reveal a similar degree of polymorphism in the numbers of V beta gene segments. We have now carried out a survey of 10 different V alpha gene segment subfamilies containing approximately 23 V alpha gene segments in a panel of 120 unrelated individuals by hybridization and failed to find any evidence for V alpha repertoire polymorphism. To determine if significant germline polymorphism does occur in humans at the level of individual V gene segments, we determined the nucleotide sequences of eight copies of the V alpha 21 gene segment derived from seven unrelated individuals. Polymorphic differences between these sequences defined three different alleles. One of these alleles contains a frameshift mutation which would cause premature termination of the protein product. The presence of this null allele among the eight sequences determined suggests that functionally relevant germline polymorphism of human TCR V gene segments may occur by mechanisms other than gene duplication or deletion.

Amino Acid Sequence↗